Antibiotic concentrations associated with antibiotic bone cements may cause skeletal cell toxicity and prevent fracture healing. We investigated toxicity effects of dose and treatment time after exposure to three antibiotics commonly used in orthopaedic local drug delivery systems. We hypothesized a threshold exists for toxicity of osteoblasts and chondrocytes after treatment with ciprofloxacin, vancomycin, or tobramycin. To test this hypothesis, we first determined whether treatment with antibiotics caused differences in cellular morphology. Cells exposed to ciprofloxacin showed considerable changes in spread, cell membrane, and extensions. We next asked what dosage of antibiotic would cause reductions in osteoblast and chondrocyte cell numbers. Ciprofloxacin at a dose greater than 100 microg/mL and vancomycin and tobramycin at doses greater than 2000 microg/mL severely decreased cellular proliferation. Finally, we questioned whether observed decreases in cell numbers were the result of increased cellular toxicity or senescence. Released lactate dehydrogenase ratios were severely increased in osteoblasts. These data suggest the balance between the targeted microbicidal effects and host cellular toxicity is critical for skeletal cell survival and function.